β2-AR-induced Her2Transactivation Mediated By Erbin Confers Protection From Apoptosis In Cardiomyocytes

Backgroundβ-adrenergic receptor (beta-AR) is an important member of the G protein-coupled receptor(GPCR) family. They mediate the stimulatory effects of in heart，converting sympathetic nervous systemsignals into cardiovascular responses to regulate contraction rate and contractility. However， the precisemechanisms underlying are not fully understood.A fundamental role for Her2in cardioprotection has been recognized. The importance of Her2innormal cardiac development and physiology was also proposed using cardiac specific Her2knockout mice.Clinical studies have addressed the cardiotoxic effects of the therapeutic antibody Herceptin targeting Her2.A recent report revealed a potential role of the interaction between Her2and β2-AR in ERK1/2activationinduced by multiple β2-AR ligand in the heart. However， the study also indicates that other unidentifiedscaffolding molecules are required for the interaction between Her2and β2-AR.Erbin is a member of the LAP (leucine-rich repeat and PDZ domain) protein family and the PDZdomain of Erbin binds to the C-terminal region of Her2. In the present study， we demonstrate that PDZdomain of Erbin scaffolds the interaction of β2-AR with Her2， promotes β2-AR-mediated ERK activationand confers protection from apoptosis induced by chronic catecholamine stimulation in cardiomyocytes.PurposesIn present study， we aim to elucidate whether Erbin serve as a scaffold protein to mediate theinteraction between Her2and β2-AR， reveal the precise mechanism， and preliminary explore thebiological functions of Erbin in cardiomyocytes.MethodsTo verify our hypothesis， that Her2， β2-AR and Erbin formed a complex， the coimmunoprecipitation assay was employed in rat heart and brain tissues. To further our hypothesis， theexogenous and endogenous coimmunoprecipitation assay were conducted in293T and rat cardiomyocytecell line H9C2cells respectively. To further confirm that the key role of Erbin in assembling β2-AR/Her2complex， the subcellular localization of Erbin in response to β2-AR agonist stimulation was detected，and the siRNA targeting Erbin was utilized. To map the β2-AR-binding region of Erbin， we created aseries of Erbin deletion mutants carried a Myc or hemagglutinin (HA) tag，and coimmunoprecipitation andconfocal immunofluorescence microscopic assays were employed. To further evaluate whethercatecholamine-induced activation of ERK depends on Erbin-mediated β2-AR/Her2complexation， COS-7cells were cotransfected with the plasmids expressing β2-AR， Her2and HA-Erbin PDZ， together withErbin siRNA. To determine the functional consequences of Erbin in cardiomyocyte apoptosis， H9c2cellswere transfected with the Erbin siRNA and then treated with ISO， and cellular apoptosis was assayed byDeadEndTM Colorimetric TUNEL System. To determine whether disruption of Erbin-mediatedβ2-AR/Her2complexation has the potential impact on the cardiomyocyte contractile function in responseto agonist-induced β2-AR activation， the primary neonatal rat cardiomyocytes were transfected with thesiRNA targeting Erbin and the frequency of spontaneous beating measured.ResultsThe data of coimmunoprecipitation assay conducted in rat heart and brain tissues confirmed ourhypothesis， as Erbin could be coimmunoprecipitated with β2-AR. Further experiments testified theexistence of β2-AR， Her2and Erbin complex， demonstrating that Erbin is a new binding partner ofβ2-AR and β2-AR/Her2complex contains Erbin. Subsequently， we proved that Erbin scaffolded theinteraction of exogenous Her2and β2-AR in293T cells， and Erbin assembled endogenous β2-AR/Her2complex in H9C2cells. Western blot analysis exhibited that Erbin in the plasma membrane fraction wasprominently increased in a time-dependent manner concomitant with the decline of Erbin protein in thecytosolic fraction， demonstrating the translocation of Erbin from the cytosol to cytoplasm membraneafter ISO stimulation. The perturbation of Erbin expression dramatically abolished the association ofβ2-AR and Her2， indicating that ISO stimulates the formation of β2-AR， Her2and Erbin complex and that Erbin scaffolds the interaction between β2-AR and Her2. Domain mapping data indicated that the PDZdomain of Erbin is required for ISO-stimulated β2-AR/Her2complexation. Cell signaling assaydemonstrated that Erbin positively promotes Her2-mediated ERK activation by β2-AR agonist incardiomyocytes， since silencing of Erbin greatly abrogated ISO-induced activation of ERK. The treatmentof H9c2cells transfected with the Erbin siRNA with ISO caused severe cell apoptosis. Knock-down ofErbin expression in primary neonatal rat cardiomyocytes led to a remarkable reduction of the beatingfrequency after ISO stimulation.ConclusionsErbin mediates catecholamine-induced β2-AR/Her2complexation and promotescatecholamine-induced activation of ERK signaling in cardiomyocytes， conferring protection ofcardiomyocytes from apoptosis induced by chronic catecholamine stimulation.